Kinesin-3 is an organelle motor in the squid giant axon

Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.     

Kinesin is bound with high affinity to squid axon organelles that move to the plus-end of microtubules

This paper addresses the question of whether microtubule-directed transport of vesicular organelles depends on the presence of a pool of cytosolic factors, including soluble motor proteins and accessory factors. Earlier studies with squid axon organelles (Schroer et al., 1988) suggested that the presence of cytosol induces a > 20-fold increase in the number of organelles moving per unit time on microtubules in vitro. These earlier studies, however, did not consider that cytosol might nonspecifically increase the numbers of moving organelles, i.e., by blocking adsorption of organelles to the coverglass. Here we report that treatment of the coverglass with casein, in the absence of cytosol, blocks adsorption of organelles to the coverglass and results in vigorous movement of vesicular organelles in the complete absence of soluble proteins. This technical improvement makes it possible, for the first time, to perform quantitative studies of organelle movement in the absence of cytosol. These new studies show that organelle movement activity (numbers of moving organelles/min/micron microtubule) of unextracted organelles is not increased by cytosol. Unextracted organelles move in single directions, approximately two thirds toward the plus-end and one third toward the minus-end of microtubules. Extraction of organelles with 600 mM KI completely inhibits minus-end, but not plus-end directed organelle movement. Upon addition of cytosol, minus-end directed movement of KI organelles is restored, while plus--end directed movement is unaffected. Biochemical studies indicate that KI-extracted organelles attach to microtubules in the presence of AMP-PNP and copurify with tightly bound kinesin. The bound kinesin is not extracted from organelles by 1 M KI, 1 M NaCl or carbonate (pH 11.3). These results suggest that kinesin is irreversibly bound to organelles that move to the plus-end of microtubules and that the presence of soluble kinesin and accessory factors is not required for movement of plus-end organelles in squid axons.     

Regulation of pigment organelle translocation. I. Phosphorylation of the organelle-associated protein p57

Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57.     

BP-80 and homologs are concentrated on post-Golgi,probable lytic prevacuolar compartments

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.     

Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2

Salmonella enterica forms polyhedral organelles during coenzyme B(12)-dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B(12)-dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B(12)-dependent 1,2-PD degradation by S. enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B(12)-dependent diol dehydratase. Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB'CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB'JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO(2) fixation), suggesting that the S. enterica organelles and carboxysomes have a related multiprotein shell. In addition, S. enterica organelles contained four enzymes: B(12)-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the S. enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S. enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.     

Dynamic shape changes of cytoplasmic organelles translocating along microtubules

Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high- resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules.     

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.     

Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo

Brain dynein is a microtubule-activated ATPase considered to be a candidate to function as a molecular motor to transport membranous organelles retrogradely in the axon. To determine whether brain dynein really binds to retrogradely transported organelles in vivo and how it is transported to the nerve terminals, we studied the localization of brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using affinity-purified anti-brain dynein antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Interestingly, brain dynein accumulated both at the regions proximal and distal to the ligation sites and localized not only on retrogradely transported membranous organelles but also on anterogradely transported ones. This is the first evidence to show that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activates brain dynein only for retrograde transport.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Association of kinesin with characterized membrane-bounded organelles.

The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.     

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.     

Mechanical properties of organelles driven by microtubule-dependent molecular motors in living cells

The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is ～ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.     

Influence of translocation track on the motion of intra-axonally transported organelles in human nerve

The mechanism by which organelles are transported bidirectionally in axoplasm is still unknown; however, evidence of a key role for microtubules in many nonmammalian models has been established. We have observed common or shared tracks within the axoplasm of human nerves along which multiple organelles of varying size and shape are bidirectionally transported. Organelles traveling anterogradely and retrogradely were visualized by video-enhanced differential interference contrast optics and analyzed with the aid of computer-image-processing techniques. Speeds of translocating organelles were determined at eight to 16 translocation points along a path or "track." Each translocation speed was plotted against its corresponding position on the track to develop a "speed/position diagram." Regardless of mean organelle speed or direction of motion, organelles sharing a common track exhibited similar patterns of "speeding up" and "slowing down" relative to position along the track. Speed position data for organelles translocating the local axonal region of a common track showed no unique patterns (not different from a uniform distribution, p less than 0.05). The unique speed/position patterns exhibited by common tracks were not necessarily related to the patterns of other tracks in the immediate vicinity (distance between tracks of less than 0.50 micron). These findings suggest that there are "common tracks" shared by organelles moving retrogradely and anterogradely; both the organelles and the "track" associated with its translocation play a role in the resultant motion of that organelle; the influence exerted by a common track on the motion of an organelle results in a pattern of speed changes related to position along the track.     

Kinesin associates with anterogradely transported membranous organelles in vivo

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.     

Lysosome-related organelles.

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.     

Kinetic analysis of the synthesis and assembly of type 1 fimbriae of Escherichia coli.

The adhesive organelles (type 1 fimbriae) of K-12 and other isolates of Escherichia coli are composed of identical 17,000-dalton subunits. We examined the assembly of these subunits into fimbrial organelles. After synthesis, the nascent subunits were first processed and then assembled into the organelles; the assembly step took almost 3 min in log-phase cultures at 37 degrees C. Even during blockage of protein synthesis, the free subunits continued to assemble until the pool was depleted. This pool was small in comparison with the amount of total fimbrial protein already assembled into surface organelles and was not sufficient to regenerate new detectable organelles after the removal of preexistent ones by blending. Assembly appeared to slow when the metabolic rate of the bacterial cells slowed, since subunits took longer to appear in the organelles at lower than optimal temperatures or as a culture entered the stationary phase. The synthetic rate of subunits slowed sooner than that of total cellular proteins as a culture approached the stationary phase and ceased completely as the culture entered the stationary phase. The amount of fimbrial antigen expressed on the surface of the cells remained relatively constant during growth of a culture.     

Structural basis of cytoplasmic streaming in Characean internodal cells. A hydrodynamic analysis

Summary Hydrodynamic equations were derived which relate the velocity profile of endoplasmic streaming with the motive force generated by active sliding of endoplasmic organelles in Characean internodal cells, under two implicit assumptions that (1) the sliding velocity of putative organelles is comparable to the streaming velocity of endoplasm, and (2) subcortical endoplasm is far less viscous than bulk endoplasm.The equations were extended so as to calculate the velocity profile in flattened or perfused internodal cells. Calculated profiles were basically consistent with reported patterns of streaming under these conditions.Utilizing published data, we deduce some hydrodynamic parameters of streaming, and predict the dimensions of putative organelles expected to drive entire cytoplasm. A revision for published values of the motive force of streaming is proposed.Hydrodynamic analyses made earlier on the spherical organelles are repeated. The results show that the organelles may generate streaming, depending on the configurationin vivo of fine filaments protruding from the body of the organelles.     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Patterns of organelle distribution in mouse embryos during preimplantation development

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.     

Signalling mechanisms involved in apical organelle discharge during host cell invasion by apicomplexan parasites

Malaria is caused by Plasmodium parasites, which belong to the phylum apicomplexa. The characteristic feature of apicomplexan parasites is the presence of apical organelles, referred to as micronemes and rhoptries, in the invasive stages of the parasite life cycle. Survival of these obligate intracellular parasites depends on successful invasion of host cells, which is mediated by specific molecular interactions between host receptors and parasite ligands that are commonly stored in these apical organelles. The timely release of these ligands from apical organelles to the parasite surface is crucial for receptor engagement and invasion. This article is a broad overview of the signalling mechanisms that control the regulated secretion of apical organelles during host cell invasion by apicomplexan parasites.